Gibberellins: Regulators of Plant Height 20 Chapter FOR NEARLY 30 YEARS after the discovery of auxin in 1927, and more than 20 years after its structural elucidation as indole-3-acetic acid, West- ern plant scientists tried to ascribe the regulation of all developmental phenomena in plants to auxin. However, as we will see in this and sub- sequent chapters, plant growth and development are regulated by sev- eral different types of hormones acting individually and in concert. In the 1950s the second group of hormones, the gibberellins (GAs), was characterized. The gibberellins are a large group of related com- pounds (more than 125 are known) that, unlike the auxins, are defined by their chemical structure rather than by their biological activity. Gib- berellins are most often associated with the promotion of stem growth, and the application of gibberellin to intact plants can induce large increases in plant height. As we will see, however, gibberellins play important roles in a variety of physiological phenomena. The biosynthesis of gibberellins is under strict genetic, developmen- tal, and environmental control, and numerous gibberellin-deficient mutants have been isolated. Mendel’s tall/dwarf alleles in peas are a famous example. Such mutants have been useful in elucidating the com- plex pathways of gibberellin biosynthesis. We begin this chapter by describing the discovery, chemical structure, and role of gibberellins in regulating various physiological processes, including seed germination, mobilization of endosperm storage reserves, shoot growth, flowering, floral development, and fruit set. We then examine biosynthesis of the gibberellins, as well as identification of the active form of the hormone. In recent years, the application of molecular genetic approaches has led to considerable progress in our understanding of the mechanism of gibberellin action at the molecular level. These advances will be dis- cussed at the end of the chapter. THE DISCOVERY OF THE GIBBERELLINS Although gibberellins did not become known to American and British scientists until the 1950s, they had been dis- covered much earlier by Japanese scientists. Rice farmers in Asia had long known of a disease that makes the rice plants grow tall but eliminates seed production. In Japan this disease was called the “foolish seedling,” or bakanae, disease. Plant pathologists investigating the disease found that the tallness of these plants was induced by a chemical secreted by a fungus that had infected the tall plants. This chemical was isolated from filtrates of the cultured fungus and called gibberellin after Gibberella fujikuroi, the name of the fungus. In the 1930s Japanese scientists succeeded in obtaining impure crystals of two fungal growth-active compounds, which they termed gibberellin A and B, but because of com- munication barriers and World War II, the information did not reach the West. Not until the mid-1950s did two groups—one at the Imperial Chemical Industries (ICI) research station at Welyn in Britain, the other at the U.S. Department of Agriculture (USDA) in Peoria, Illinois—suc- ceed in elucidating the structure of the material that they had purified from fungal culture filtrates, which they named gibberellic acid: At about the same time scientists at Tokyo University isolated three gibberellins from the original gibberellin A and named them gibberellin A 1 , gibberellin A 2 , and gib- berellin A 3 . Gibberellin A 3 and gibberellic acid proved to be identical. It became evident that an entire family of gibberellins exists and that in each fungal culture different gibberellins predominate, though gibberellic acid is always a principal component. As we will see, the structural feature that all gibberellins have in common, and that defines them as a family of molecules, is that they are derived from the ent- kaurene ring structure: As gibberellic acid became available, physiologists began testing it on a wide variety of plants. Spectacular responses were obtained in the elongation growth of dwarf and rosette plants, particularly in genetically dwarf peas ( Pisum sativum ), dwarf maize (Zea mays), and many rosette plants. In contrast, plants that were genetically very tall showed no further response to applied gibberellins. More recently, experiments with dwarf peas and dwarf corn have con- firmed that the natural elongation growth of plants is reg- ulated by gibberellins, as we will describe later. Because applications of gibberellins could increase the height of dwarf plants, it was natural to ask whether plants contain their own gibberellins. Shortly after the discovery of the growth effects of gibberellic acid, gibberellin-like substances were isolated from several species of plants. 1 Gibberellin-like substance refers to a compound or an extract that has gibberellin-like biological activity, but whose chemical structure has not yet been defined. Such a response indicates, but does not prove, that the tested sub- stance is a gibberellin. In 1958 a gibberellin (gibberellin A 1 ) was conclusively identified from a higher plant (runner bean seeds, Phaseo- lus coccineus ): Because the concentration of gibberellins in immature seeds far exceeds that in vegetative tissue, immature seeds were the tissue of choice for gibberellin extraction. However, because the concentration of gibberellins in plants is very low (usually 1–10 parts per billion for the active gibberellin in vegetative tissue and up to 1 part per million of total gib- berellins in seeds), chemists had to use truckloads of seeds. As more and more gibberellins from fungal and plant sources were characterized, they were numbered as gib- berellin A X (or GA X ), where X is a number, in the order of their discovery. This scheme was adopted for all gib- berellins in 1968. However, the number of a gibberellin is simply a cataloging convenience, designed to prevent chaos in the naming of the gibberellins. The system implies no close chemical similarity or metabolic relationship between gibberellins with adjacent numbers. All gibberellins are based on the ent-gibberellane skeleton: 2 3 1 4 18 19 15 13 1211 16 17 10 20 5 6 7 8 H H A B 9 C 14 D ent-Gibberellane structure COOH O CO CH 3 H CH 2 HO OH Gibberellin A 1 (GA 1 ) CH 2 ent-Kaurene COOH O CO CH 3 H CH 2 HO OH Gibberellic acid (GA 3 ) 1 Phinney (1983) provides a wonderful personal account of the history of gibberellin discoveries. 462 Chapter 20 Some gibberellins have the full complement of 20 carbons (C 20 -GAs): Others have only 19 (C 19 -GAs), having lost one carbon to metabolism. There are other variations in the basic structure, espe- cially the oxidation state of carbon 20 (in C 20 -GAs) and the number and position of hydroxyl groups on the molecule (see Web Topic 20.1). Despite the plethora of gibberellins present in plants, genetic analyses have demonstrated that only a few are biologically active as hormones. All the oth- ers serve as precursors or represent inactivated forms. EFFECTS OF GIBBERELLIN ON GROWTH AND DEVELOPMENT Though they were originally discovered as the cause of a disease of rice that stimulated internode elongation, endogenous gibberellins influence a wide variety of devel- opmental processes. In addition to stem elongation, gib- berellins control various aspects of seed germination, including the loss of dormancy and the mobilization of endosperm reserves. In reproductive development, gib- berellin can affect the transition from the juvenile to the mature stage, as well as floral initiation, sex determination, and fruit set. In this section we will review some of these gibberellin-regulated phenomena. Gibberellins Stimulate Stem Growth in Dwarf and Rosette Plants Applied gibberellin promotes internodal elongation in a wide range of species. However, the most dramatic stimu- lations are seen in dwarf and rosette species, as well as members of the grass family. Exogenous GA 3 causes such extreme stem elongation in dwarf plants that they resem- ble the tallest varieties of the same species (Figure 20.1). Accompanying this effect are a decrease in stem thickness, a decrease in leaf size, and a pale green color of the leaves. Some plants assume a rosette form in short days and undergo shoot elongation and flowering only in long days (see Chapter 24). Gibberellin application results in bolting (stem growth) in plants kept in short days (Figure 20.2), and normal bolting is regulated by endogenous gibberellin. In addition, as noted earlier, many long-day rosette plants have a cold requirement for stem elongation and flower- ing, and this requirement is overcome by applied gib- berellin. GA also promotes internodal elongation in members of the grass family. The target of gibberellin action is the inter- calary meristem —a meristem near the base of the intern- ode that produces derivatives above and below. Deep- water rice is a particularly striking example. We will examine the effects of gibberellin on the growth of deep- water rice in the section on the mechanism of gibberellin- induced stem elongation later in the chapter. Although stem growth may be dramatically enhanced by GAs, gibberellins have little direct effect on root growth. However, the root growth of extreme dwarfs is less than that of wild-type plants, and gibberellin application to the shoot enhances both shoot and root growth. Whether the effect of gibberellin on root growth is direct or indirect is currently unresolved. Gibberellins Regulate the Transition from Juvenile to Adult Phases Many woody perennials do not flower until they reach a certain stage of maturity; up to that stage they are said to H 3 C COOH COOH H 3 C 6 20 7 H H CH 2 GA 12 (a C 20 -gibberellin) FIGURE 20.1 The effect of exogenous GA 1 on normal and dwarf ( d1) corn. Gibberellin stimulates dramatic stem elon- gation in the dwarf mutant but has little or no effect on the tall wild-type plant. (Courtesy of B. Phinney.) Gibberellins: Regulators of Plant Height 463 be juvenile (see Chapter 24). The juvenile and mature stages often have different leaf forms, as in English ivy ( Hedera helix) (see Figure 24.9). Applied gibberellins can regulate this juvenility in both directions, depending on the species. Thus, in English ivy GA 3 can cause a reversion from a mature to a juvenile state, and many juvenile conifers can be induced to enter the reproductive phase by applications of nonpolar gibberellins such as GA 4 + GA 7 . (The latter example is one instance in which GA 3 is not effective.) Gibberellins Influence Floral Initiation and Sex Determination As already noted, gibberellin can substitute for the long- day or cold requirement for flowering in many plants, especially rosette species (see Chapter 24). Gibberellin is thus a component of the flowering stimulus in some plants, but apparently not in others. In plants where flowers are unisexual rather than her- maphroditic, floral sex determination is genetically regu- lated. However, it is also influenced by environmental fac- tors, such as photoperiod and nutritional status, and these environmental effects may be mediated by gibberellin. In maize, for example, the staminate flowers (male) are restricted to the tassel, and the pistillate flowers (female) are contained in the ear. Exposure to short days and cool nights increases the endogenous gibberellin levels in the tassels 100-fold and simultaneously causes feminization of the tassel flowers. Application of exogenous gibberellic acid to the tassels can also induce pistillate flowers. For studies on genetic regulation, a large collection of maize mutants that have altered patterns of sex determi- nation have been isolated. Mutations in genes that affect either gibberellin biosynthesis or gibberellin signal trans- duction result in a failure to suppress stamen development in the flowers of the ear (Figure 20.3). Thus the primary role of gibberellin in sex determination in maize seems to be to suppress stamen development (Irish 1996). In dicots such as cucumber, hemp, and spinach, gib- berellin seems to have the opposite effect. In these species, application of gibberellin promotes the formation of sta- minate flowers, and inhibitors of gibberellin biosynthesis promote the formation of pistillate flowers. Gibberellins Promote Fruit Set Applications of gibberellins can cause fruit set (the initia- tion of fruit growth following pollination) and growth of some fruits, in cases where auxin may have no effect. For example, stimulation of fruit set by gibberellin has been observed in apple ( Malus sylvestris). Gibberellins Promote Seed Germination Seed germination may require gibberellins for one of sev- eral possible steps: the activation of vegetative growth of FIGURE 20.2 Cabbage, a long-day plant, remains as a rosette in short days, but it can be induced to bolt and flower by applications of gibberellin. In the case illustrated, giant flowering stalks were produced. (© Sylvan Wittwer/Visuals Unlimited.) FIGURE 20.3 Anthers develop in the ears of a gibberellin- deficient dwarf mutant of corn ( Zea mays). (Bottom) Unfertilized ear of the dwarf mutant an1, showing conspic- uous anthers. (Top) Ear from a plant that has been treated with gibberellin. (Courtesy of M. G. Neuffer.) 464 Chapter 20 the embryo, the weakening of a growth-constraining endosperm layer surrounding the embryo, and the mobi- lization of stored food reserves of the endosperm. Some seeds, particularly those of wild plants, require light or cold to induce germination. In such seeds this dormancy (see Chapter 23) can often be overcome by application of gib- berellin. Since changes in gibberellin levels are often, but not always, seen in response to chilling of seeds, gib- berellins may represent a natural regulator of one or more of the processes involved in germination. Gibberellin application also stimulates the production of numerous hydrolases, notably α-amylase, by the aleu- rone layers of germinating cereal grains. This aspect of gib- berellin action has led to its use in the brewing industry in the production of malt (discussed in the next section). Because this is the principal system in which gibberellin signal transduction pathways have been analyzed, it will be treated in detail later in the chapter. Gibberellins Have Commercial Applications The major uses of gibberellins (GA 3 , unless noted other- wise), applied as a spray or dip, are to manage fruit crops, to malt barley, and to increase sugar yield in sugarcane. In some crops a reduction in height is desirable, and this can be accomplished by the use of gibberellin synthesis inhibitors (see Web Topic 20.1). Fruit production. A major use of gibberellins is to increase the stalk length of seedless grapes. Because of the shortness of the individual fruit stalks, bunches of seedless grapes are too compact and the growth of the berries is restricted. Gib- berellin stimulates the stalks to grow longer, thereby allow- ing the grapes to grow larger by alleviating compaction, and it promotes elongation of the fruit (Figure 20.4). A mixture of benzyladenine (a cytokinin; see Chapter 21) and GA 4 + GA 7 can cause apple fruit to elongate and is used to improve the shape of Delicious-type apples under certain conditions. Although this treatment does not affect yield or taste, it is considered commercially desirable. In citrus fruits, gibberellins delay senescence, allowing the fruits to be left on the tree longer to extend the market period. Malting of barley. Malting is the first step in the brew- ing process. During malting, barley seeds ( Hordeum vulgare) are allowed to germinate at temperatures that maximize the production of hydrolytic enzymes by the aleurone layer. Gibberellin is sometimes used to speed up the malt- ing process. The germinated seeds are then dried and pul- verized to produce “malt,” consisting mainly of a mixture of amylolytic (starch-degrading) enzymes and partly digested starch. During the subsequent “mashing” step, water is added and the amylases in the malt convert the residual starch, as well as added starch, to the disaccharide maltose, which is converted to glucose by the enzyme maltase. The resulting “wort” is then boiled to stop the reaction. In the final step, yeast converts the glucose in the wort to ethanol by fer- mentation. Increasing sugarcane yields. Sugarcane (Saccharum offic- inarum ) is one of relatively few plants that store their car- bohydrate as sugar (sucrose) instead of starch (the other important sugar-storing crop is sugar beet). Originally from New Guinea, sugarcane is a giant perennial grass that can grow from 4 to 6 m tall. The sucrose is stored in the central vacuoles of the internode parenchyma cells. Spraying the crop with gibberellin can increase the yield of raw cane by up to 20 tons per acre, and the sugar yield by 2 tons per acre. This increase is a result of the stimulation of internode elongation during the winter season. Uses in plant breeding. The long juvenility period in conifers can be detrimental to a breeding program by pre- venting the reproduction of desirable trees for many years. Spraying with GA 4 + GA 7 can considerably reduce the time to seed production by inducing cones to form on very young trees. In addition, the promotion of male flowers in cucurbits, and the stimulation of bolting in biennial rosette crops such as beet ( Beta vulgaris) and cabbage (Brassica oler- acea ), are beneficial effects of gibberellins that are occa- sionally used commercially in seed production. Gibberellin biosynthesis inhibitors. Bigger is not always better. Thus, gibberellin biosynthesis inhibitors are used commercially to prevent elongation growth in some plants. In floral crops, short, stocky plants such as lilies, chrysan- themums, and poinsettias are desirable, and restrictions on elongation growth can be achieved by applications of gib- berellin synthesis inhibitors such as ancymidol (known commercially as A-Rest) or paclobutrazol (known as Bonzi). FIGURE 20.4 Gibberellin induces growth in Thompson’s seedless grapes. The bunch on the left is an untreated con- trol. The bunch on the right was sprayed with gibberellin during fruit development. (© Sylvan Wittwer/Visuals Unlimited.) Gibberellins: Regulators of Plant Height 465 Tallness is also a disadvantage for cereal crops grown in cool, damp climates, as occur in Europe, where lodging can be a problem. Lodging—the bending of stems to the ground caused by the weight of water collecting on the ripened heads—makes it difficult to harvest the grain with a com- bine harvester. Shorter internodes reduce the tendency of the plants to lodge, increasing the yield of the crop. Even genetically dwarf wheats grown in Europe are sprayed with gibberellin biosynthesis inhibitors to further reduce stem length and lodging. Yet another application of gibberellin biosynthesis inhibitors is the restriction of growth in roadside shrub plantings. BIOSYNTHESIS AND METABOLISM OF GIBBERELLIN Gibberellins constitute a large family of diterpene acids and are synthesized by a branch of the terpenoid pathway, which was described in Chapter 13. The elucidation of the gibberellin biosynthetic pathway would not have been pos- sible without the development of sensitive methods of detection. As noted earlier, plants contain a bewildering array of gibberellins, many of which are biologically inactive. In this section we will discuss the biosynthesis of GAs, as well as other factors that regulate the steady-state levels of the biologically active form of the hormone in different plant tissues. Gibberellins Are Measured via Highly Sensitive Physical Techniques Systems of measurement using a biological response, called bioassays, were originally important for detecting gib- berellin-like activity in partly purified extracts and for assessing the biological activity of known gibberellins (Fig- ure 20.5). The use of bioassays, however, has declined with the development of highly sensitive physical techniques that allow precise identification and quantification of spe- cific gibberellins from small amounts of tissue. High-performance liquid chromatography (HPLC) of plant extracts, followed by the highly sensitive and selec- tive analytical method of gas chromatography combined with mass spectrometry (GC-MS), has now become the method of choice. With the availability of published mass spectra, researchers can now identify gibberellins without possessing pure standards. The availability of heavy-iso- tope-labeled standards of common gibberellins, which can themselves be separately detected on a mass spectrometer, allows the accurate measurement of levels in plant tissues by mass spectrometry with these heavy-isotope-labeled gibberellins as internal standards for quantification (see Web Topic 20.2). Gibberellins Are Synthesized via the Terpenoid Pathway in Three Stages Gibberellins are tetracyclic diterpenoids made up of four isoprenoid units. Terpenoids are compounds made up of five-carbon (isoprene) building blocks: joined head to tail. Researchers have determined the entire gibberellin biosynthetic pathway in seed and vegetative tis- sues of several species by feeding various radioactive pre- cursors and intermediates and examining the production of the other compounds of the pathway (Kobayashi et al. 1996). The gibberellin biosynthetic pathway can be divided into three stages, each residing in a different cellular com- partment (Figure 20.6) (Hedden and Phillips 2000). C CH 2 OH CH CH 2 FIGURE 20.5 Gibberellin causes elongation of the leaf sheath of rice seedlings, and this response is used in the dwarf rice leaf sheath bioassay. Here 4-day-old seedlings were treated with dif- ferent amounts of GA and allowed to grow for another 5 days. (Courtesy of P. Davies.) 466 Chapter 20 OPP OPP COOH COOH OH COOH COOH R COOH COOH COOH COOH HOCH 2 R COOH O HO CO R COOH O CO R COOH O HO CO R COOH O HO HO CO R COOH COOH CHO R ent-Kaurene ent-Kaurene GA 12 -aldehyde ent-Copalyl diphosphate GGPP COOHCH 3 CH 3 CHO GA 12 GA 53 GA 12 (R = H) GA 53 (R = OH) GA 20-oxidase GA 2-oxidaseGA 2-oxidase GA 15 -OL (R = H) GA 44 -OL (R = OH) GA 20-oxidase GA 20-oxidase GA 3-oxidase Active GA GA 4 (R = H) GA 1 (R = OH) GA 9 (R = H) GA 20 (R = OH) GA 34 (R = H) GA 8 (R = OH) GA 51 (R = H) GA 29 (R = OH) GA 24 (R = H) GA 19 (R = OH) PLASTID ENDOPLASMIC RETICULUM CYTOSOL Stage 1 Stage 2 Stage 3 Inactivation FIGURE 20.6 The three stages of gibberellin biosynthesis. In stage 1, geranylgeranyl diphosphate (GGPP) is converted to ent-kaurene via copalyl diphosphate (CPP) in plastids. In stage 2, which takes place on the endoplasmic reticulum, ent-kaurene is converted to GA 12 or GA 53 , depending on whether the GA is hydroxylated at carbon 13. In most plants the 13-hydroxylation pathway predominates, though in Arabidopsis and some others the non-13-OH pathway is the main pathway. In stage 3 in the cytosol, GA 12 or GA 53 are converted other GAs. This conversion proceeds with a series of oxidations at carbon 20. In the 13-hydroxylation pathway this leads to the production of GA 20 . GA 20 is then oxidized to the active gibberellin, GA 1 , by a 3β-hydroxyla- tion reaction (the non-13-OH equivalent is GA 4 ). Finally, hydroxylation at carbon 2 converts GA 20 and GA 1 to the inactive forms GA 29 and GA 8 , respectively. Stage 1: Production of terpenoid precursors and ent-kau- rene in plastids. The basic biological isoprene unit is isopentenyl diphosphate (IPP). 2 IPP used in gibberellin biosynthesis in green tissues is synthesized in plastids from glyceraldehyde-3-phosphate and pyruvate (Lichtenthaler et al. 1997). However, in the endosperm of pumpkin seeds, which are very rich in gibberellin, IPP is formed in the cytosol from mevalonic acid, which is itself derived from acetyl-CoA. Thus the IPP used to make gibberellins may arise from dif- ferent cellular compartments in different tissues. Once synthesized, the IPP isoprene units are added suc- cessively to produce intermediates of 10 carbons (geranyl diphosphate), 15 carbons (farnesyl diphosphate), and 20 carbons (geranylgeranyl diphosphate, GGPP). GGPP is a precursor of many terpenoid compounds, including carotenoids and many essential oils, and it is only after GGPP that the pathway becomes specific for gibberellins. The cyclization reactions that convert GGPP to ent-kau- rene represent the first step that is specific for the gib- berellins (Figure 20.7). The two enzymes that catalyze the reactions are localized in the proplastids of meristematic shoot tissues, and they are not present in mature chloro- plasts (Aach et al. 1997). Thus, leaves lose their ability to synthesize gibberellins from IPP once their chloroplasts mature. Compounds such as AMO-1618, Cycocel, and Phosphon D are specific inhibitors of the first stage of gibberellin biosynthesis, and they are used as growth height reducers. Stage 2: Oxidation reactions on the ER form GA 12 and GA 53 . In the second stage of gibberellin biosynthesis, a methyl group on ent-kaurene is oxidized to a carboxylic acid, followed by contraction of the B ring from a six- to a five-carbon ring to give GA 12 -aldehyde. GA 12 -aldehyde is then oxidized to GA 12 , the first gibberellin in the pathway in all plants and thus the precursor of all the other gib- berellins (see Figure 20.6). Many gibberellins in plants are also hydroxylated on carbon 13. The hydroxylation of carbon 13 occurs next, forming GA 53 from GA 12 . All the enzymes involved are monooxygenases that utilize cytochrome P450 in their reac- tions. These P450 monooxygenases are localized on the endoplasmic reticulum. Kaurene is transported from the plastid to the endoplasmic reticulum, and is oxidized en route to kaurenoic acid by kaurene oxidase, which is asso- ciated with the plastid envelope (Helliwell et al. 2001). Further conversions to GA 12 take place on the endo- plasmic reticulum. Paclobutrazol and other inhibitors of P450 monooxygenases specifically inhibit this stage of gib- berellin biosynthesis before GA 12 -aldehyde, and they are also growth retardants. Stage 3: Formation in the cytosol of all other gib- berellins from GA 12 or GA 53 . All subsequent steps in the pathway (see Figure 20.6) are carried out by a group of sol- uble dioxygenases in the cytosol. These enzymes require 2- oxoglutarate and molecular oxygen as cosubstrates, and they use Fe 2+ and ascorbate as cofactors. The specific steps in the modification of GA 12 vary from species to species, and between organs of the same species. Two basic chemical changes occur in most plants: 1. Hydroxylation at carbon 13 (on the endoplasmic retic- ulum) or carbon 3, or both. 2. A successive oxidation at carbon 20 (CH 2 → CH 2 OH → CHO). The final step of this oxidation is the loss of carbon 20 as CO 2 (see Figure 20.6). When these reactions involve gibberellins initially hydroxylated at C-13, the resulting gibberellin is GA 20 . GA 20 is then converted to the biologically active form, Geranylgeranyl diphosphate ls Copalyl diphosphate ent-Kaurene na slnle GA 12 -aldehyde GA 12 GA 53 GA 20-oxidase GA 44 GA 20-oxidase GA 19 GA 20-oxidase GA 2-oxidase GA 20 GA 2-oxidaseGA 3-oxidase GA 29 GA 1 GA 8 sln FIGURE 20.7 A portion of the gibberellin biosynthetic path- way showing the abbreviations and location of the mutant genes that block the pathway in pea and the enzymes involved in the metabolic steps after GA 53 . 2 As noted in Chapter 13, IPP is the abbreviation for isopen- tenyl pyrophosphate, an earlier name for this compound. Similarly, the other pyrophosphorylated intermediates in the pathway are now referred to as diphosphates, but they continue to be abbreviated as if they were called pyrophos- phates. 468 Chapter 20 GA 1 , by hydroxylation of carbon 3. (Because this is in the beta configuration [drawn as if the bond to the hydroxyl group were toward the viewer], it is referred to as 3 β- hydroxylation.) Finally, GA 1 is inactivated by its conversion to GA 8 by a hydroxylation on carbon 2. This hydroxylation can also remove GA 20 from the biosynthetic pathway by converting it to GA 29 . Inhibitors of the third stage of the gibberellin biosyn- thetic pathway interfere with enzymes that utilize 2-oxog- lutarate as cosubstrates. Among these, the compound pro- hexadione (BX-112), is especially useful because it specifically inhibits GA 3-oxidase, the enzyme that converts inactive GA 20 to growth-active GA 1 . The Enzymes and Genes of the Gibberellin Biosynthetic Pathway Have Been Characterized The enzymes of the gibberellin biosynthetic pathway are now known, and the genes for many of these enzymes have been isolated and characterized (see Figure 20.7). Most notable from a regulatory standpoint are two biosyn- thetic enzymes—GA 20-oxidase (GA20ox) 3 and GA 3-oxi- dase (GA3ox)—and an enzyme involved in gibberellin metabolism, GA 2-oxidase (GA2ox): • GA 20-oxidase catalyzes all the reactions involving the successive oxidation steps of carbon 20 between GA 53 and GA 20 , including the removal of C-20 as CO 2 . • GA 3-oxidase functions as a 3β-hydroxylase, adding a hydroxyl group to C-3 to form the active gib- berellin, GA 1 . (The evidence demonstrating that GA 1 is the active gibberellin will be discussed shortly.) • GA 2-oxidase inactivates GA 1 by catalyzing the addi- tion of a hydroxyl group to C-2. The transcription of the genes for the two gibberellin biosynthetic enzymes, as well as for GA 2-oxidase, is highly regulated. All three of these genes have sequences in com- mon with each other and with other enzymes utilizing 2- oxoglutarate and Fe 2+ as cofactors. The common sequences represent the binding sites for 2-oxoglutarate and Fe 2+ . Gibberellins May Be Covalently Linked to Sugars Although active gibberellins are free, a variety of gibberellin glycosides are formed by a covalent linkage between gibberellin and a sugar. These gibberellin conjugates are particularly prevalent in some seeds. The conjugating sugar is usually glucose, and it may be attached to the gibberellin via a car- boxyl group forming a gibberellin glycoside, or via a hydroxyl group forming a gibberellin glycosyl ether. When gibberellins are applied to a plant, a certain pro- portion usually becomes glycosylated. Glycosylation there- fore represents another form of inactivation. In some cases, applied glucosides are metabolized back to free GAs, so glucosides may also be a storage form of gibberellins (Schneider and Schmidt 1990). GA 1 Is the Biologically Active Gibberellin Controlling Stem Growth Knowledge of biosynthetic pathways for gibberellins reveals where and how dwarf mutations act. Although it had long been assumed that gibberellins were natural growth regula- tors because gibberellin application caused dwarf plants to grow tall, direct evidence was initially lacking. In the early 1980s it was demonstrated that tall stems do contain more bioactive gibberellin than dwarf stems have, and that the level of the endogenous bioactive gibberellin mediates the genetic control of tallness (Reid and Howell 1995). The gibberellins of tall pea plants containing the homozygous Le allele (wild type) were compared with dwarf plants having the same genetic makeup, except con- taining the le allele (mutant). Le and le are the two alleles of the gene that regulates tallness in peas, the genetic trait first investigated by Gregor Mendel in his pioneering study in 1866. We now know that tall peas contain much more bioac- tive GA 1 than dwarf peas have (Ingram et al. 1983). As we have seen, the precursor of GA 1 in higher plants is GA 20 (GA 1 is 3β-OH GA 20 ). If GA 20 is applied to homozy- gous dwarf ( le) pea plants, they fail to respond, although they do respond to applied GA 1 . The implication is that the Le gene enables the plants to convert GA 20 to GA 1 . Metabolic studies using both stable and radioactive isotopes demon- strated conclusively that the Le gene encodes an enzyme that 3 β-hydroxylates GA 20 to produce GA 1 (Figure 20.8). Mendel’s Le gene was isolated, and the recessive le allele was shown to have a single base change leading to a defec- tive enzyme only one-twentieth as active as the wild-type 3 GA 20-oxidase means an enzyme that oxidizes at carbon 20; it is not the same as GA 20 , which is gib- berellin 20 in the GA numbering scheme. HO OH H CH 3 CH 2 H COOH O CO OH H CH 3 CH 2 H COOH O CO + OH GA 3b-hydroxylase GA 20 GA 1 FIGURE 20.8 Conversion of GA 20 to GA 1 by GA 3β-hydroxylase, which adds a hydroxyl group (OH) to carbon 3 of GA 20 . Gibberellins: Regulators of Plant Height 469 enzyme, so much less GA 1 is produced and the plants are dwarf (Lester et al. 1997). Endogenous GA 1 Levels Are Correlated with Tallness Although the shoots of gibberellin-deficient le dwarf peas are much shorter than those of normal plants (internodes of 3 cm in mature dwarf plants versus 15 cm in mature normal plants), the mutation is “leaky” (i.e., the mutated gene pro- duces a partially active enzyme) and some endogenous GA 1 remains to cause growth. Different le alleles give rise to peas differing in their height, and the height of the plant has been correlated with the amount of endogenous GA 1 (Figure 20.9). There is also an extreme dwarf mutant of pea that has even fewer gibberellins. This dwarf has the allele na (the wild-type allele is Na), which completely blocks gibberellin biosynthesis between ent-kaurene and GA 12 -aldehyde (Reid and Howell 1995). As a result, homozygous ( nana) mutants, which are almost completely free of gibberellins, achieve a stature of only about 1 cm at maturity (Figure 20.10). However, nana plants may still possess an active GA 3β- hydroxylase encoded by Le, and thus can convert GA 20 to GA 1 . If a nana naLe shoot is grafted onto a dwarf le plant, the resulting plant is tall because the nana shoot tip can convert the GA 20 from the dwarf into GA 1 . Such observations have led to the conclusion that GA 1 is the biologically active gibberellin that regulates tallness in peas (Ingram et al. 1986; Davies 1995). The same result has been obtained for maize, a monocot, in parallel studies using genotypes that have blocks in the gibberellin biosyn- thetic pathway. Thus the control of stem elongation by GA 1 appears to be universal. Although GA 1 appears to be the primary active gib- berellin in stem growth for most species, a few other gib- 16 12 8 4 0.01 0.1 1.0 Length between nodes 4 and 6 (cm) GA 1 content of pea plants possessing three different Le le alleles le-2 le-1 Le Endogenous GA 1 (ng per plant) FIGURE 20.9 Stem elongation corresponds closely to the level of GA 1 . Here the GA 1 content in peas with three dif- ferent alleles at the Le locus is plotted against the internode elongation in plants with those alleles. The allele le-2 is a more intense dwarfing allele of Le than is the regular le-1 allele. There is a close correlation between the GA level and internode elongation. (After Ross et al. 1989.) FIGURE 20.10 Phenotypes and genotypes of peas that differ in the gibberellin content of their vegetative tissue. (All alleles are homozygous.) (After Davies 1995.) Ultradwarf: no GAs nana Dwarf: contains GA 20 Na le Tall: contains GA 1 Na Le Ultratall: contains no GAs na la cry s 470 Chapter 20 [...]... 474 Chapter 20 (A) AMO-1618 (B) BX-112 In contrast, BX-112, which blocks the conversion of GA20 to GA1, inhibits growth even in the presence of GA20 AMO-1618, which blocks GA biosynthesis at the cyclization step, does not inhibit growth in the presence of either GA20 or GA1 Control AMO-1618 AMO-1618 + GA20 AMO-1618 + GA1 40 Stem length (cm) Stem length (cm) 40 30 20 30 20 10 10 0 Control BX-112 BX-112... structures of various gibberellins and inhibitors of gibberellin biosynthesis are presented 20. 2 Gibberellin Detection Gibberellin quantitation is now routine thanks to sensitive modern physical methods of detection Gibberellins: Regulators of Plant Height 20. 3 Gibberellin-Induced Stem Elongation Various mechanisms of gibberellin-induced cell wall loosening are discussed 20. 4 CDKs and Gibberellin-Induced... Additional information on the mechanism of gibberellin regulation of the cell cycle is given 20. 5 Gibberellin-Induction of α-amylase mRNA Evidence is provided for gibberellin-induced transcription of α-amylase mRNA 20. 6 Promoter Elements and Gibberellin Responsiveness Gibberellin response elements mediate the effects of gibberellin on α-amylase transcription 20. 7 Regulation of α-amylase Gene Expression by Transcription... transcription of GA2ox (Figure 20. 19) In the absence of auxin the reverse occurs Thus the apical bud promotes growth not only through the direct biosynthesis of auxin, but also through the auxin-induced biosynthesis of GA1 (Figure 20. 20) (Ross et al 200 0; Ross and O’Neill 200 1) Figure 20. 21 summarizes some of the factors that modulate the active gibberellin level through regulation of the transcription of the... 100 GA-MYB mRNA 75 50 a-Amylase mRNA 25 0 3 6 12 18 24 Hours after exposure to GA FIGURE 20. 35 Time course for the induction of GA-MYB and α-amylase mRNA by gibberellic acid The production of GA-MYB mRNA precedes α-amylase mRNA by about 5 hours This result is consistent with the role of GA-MYB as an early GA response gene that regulates the transcription of the gene for α-amylase In the absence of GA,... gibberellin sensitivity of pea seedlings falls rapidly upon transfer from darkness to light, so the elongation rate of plants in the light is lower than in the dark, even though their total GA1 content is higher (After O’Neill et al 200 0.) Gibberellins: Regulators of Plant Height another inhibitor, BX-112, which blocks the production of GA1 from GA20, can be overcome only by GA1 (Figure 20. 16B) This result... its form (see Chapter 17)—a process referred to as de-etiolation One of the most strik- (A) ing changes is a decrease in the rate of stem elongation such that the stem in the light is shorter than the one in the dark Initially it was assumed that the light-grown plants would contain less GA1 than dark-grown plants However, light-grown plants turned out to contain more GA1 than dark-grown plants—indicating... Jacobsen, J V (1995) Gibberellin-regulated expression of a myb gene in barley aleurone cells: Evidence of myb transactivation of a high-pl alpha-amylase gene promoter Plant Cell 7: 1879–1891 Hazebroek, J P., and Metzger, J D (1990) Thermoinductive regulation of gibberellin metabolism in Thlaspi arvense L I Metabolism of [2H]-ent-Kaurenoic acid and [14C]gibberellin A12-aldehyde Plant Physiol 94: 157–165 Hedden,... production at the level of gene transcription (Jacobsen et al 1995) The two main lines of evidence were as follows: 1 GA3-stimulated α-amylase production was shown to be blocked by inhibitors of transcription and translation 2 Heavy-isotope- and radioactive-isotope-labeling studies demonstrated that the stimulation of α-amylase activity by gibberellin involved de novo synthesis of the enzyme from amino... activation of one or more preexisting transcription factors The activation of transcription factors is typically mediated by protein phosphorylation events occurring at the end of a signal transduction pathway We will now examine what is known about the signaling pathways involved in gibberellin-induced α-amylase production up to the point of GA-MYB production Gibberellins: Regulators of Plant Height . diphosphate ent-Kaurene na slnle GA 12 -aldehyde GA 12 GA 53 GA 2 0- oxidase GA 44 GA 2 0- oxidase GA 19 GA 2 0- oxidase GA 2-oxidase GA 20 GA 2-oxidaseGA 3-oxidase GA 29 GA 1 GA 8 sln FIGURE. Conversion of GA 20 to GA 1 by GA 3β-hydroxylase, which adds a hydroxyl group (OH) to carbon 3 of GA 20 . Gibberellins: Regulators of Plant Height 469 enzyme,